Recombinant vaccines have been developed with the progress of recombinant DNA technology, allowing the modification of viral genomes to produce modified viruses. In this manner, it has been possible to introduce genetic sequences into non-pathogenic viruses, so that they encode immunogenic proteins to be expressed in target cells upon infection or transduction, in order to develop a specific immune response in their host.
Such vaccines constitute a major advance in vaccine technology (Kutzler et al., Nat Rev Genet, 9(10): 776-788, 2008). In particular, they have the advantage over traditional vaccines of avoiding live (attenuated) virus and eliminating risks associated with the manufacture of inactivated vaccines.
Gene delivery using modified retroviruses (retroviral vectors) was introduced in the early 1980s by Mann et al. (Cell, 33(1):153-9, 1983). The most commonly used oncogenic retroviral vectors are based on the Moloney murine leukemia virus (MLV). They have a simple genome from which the polyproteins Gag, Pol and Env are produced and are required in trans for viral replication (Breckpot et al., 2007, Gene Ther, 14(11):847-62; He et al. 2007, Expert Rev vaccines, 6(6):913-24). Sequences generally required in cis are the long terminal repeats (LTRs) and its vicinity: the inverted repeats (IR or att sites) required for integration, the packaging sequence ψ the transport RNA-binding site (primer binding site, PBS), and some additional sequences involved in reverse transcription (the repeat R within the LTRs, and the polypurine tracts, PPT, necessary for plus strand initiation). To generate replication-defective retroviral vectors, the gag, pol, and env genes are generally entirely deleted and replaced with an expression cassette.
Retroviral vectors deriving from lentivirus genomes (i.e. lentiviral vectors) have emerged as promising tools for both gene therapy and immunotherapy purposes, because they exhibit several advantages over other viral systems. In particular, lentiviral vectors themselves are not toxic and, unlike other retroviruses, lentiviruses are capable of transducing non-dividing cells, in particular dendritic cells (He et al. 2007, Expert Rev vaccines, 6(6):913-24), allowing antigen presentation through the endogenous pathway.
Lentiviruses are linked by similarities in genetic composition, molecular mechanisms of replication and biological interactions with their hosts. They are best known as agents of slow disease syndromes that begin insidiously after prolonged periods of subclinical infection and progress slowly; thus, they are referred to as the “slow” viruses (Narayan et al., 1989, J Gen Virol, 70(7):1617-39). They have the same basic organization as all retroviruses but are more complex due to the presence of accessory genes (e.g., vif, vpr, vpu, nef, tat, and rev), which play key roles in lentiviral replication in vivo.
Lentiviruses represent a genus of slow viruses of the Retroviridae family, which includes the human immunodeficiency viruses (HIV), the simian immunodeficiency virus (SIV), the equine infectious encephalitis virus (EIAV), the caprine arthritis encephalitis virus (CAEV), the bovine immunodeficiency virus (BIV) and the feline immunodeficiency virus (FIV). Lentiviruses can persist indefinitely in their hosts and replicate continuously at variable rates during the course of the lifelong infection. Persistent replication of the viruses in their hosts depends on their ability to circumvent host defenses.
The design of recombinant integrating lentiviral vectors is based on the separation of the cis- and trans-acting sequences of the lentivirus. Efficient transduction in non-dividing cells requires the presence of two cis-acting sequences in the lentiviral genome, the central polypurine tract (cPPT) and the central termination sequence (CTS). These lead to the formation of a triple-stranded DNA structure called the central DNA “flap”, which maximizes the efficiency of gene import into the nuclei of non-dividing cells, including dendritic cells (DCs) (Zennou et al., 2000, Cell, 101(2) 173-85; Arhel et al., 2007, EMBO J, 26(12):3025-37).
Dendritic cells are of primary importance for antigen presentation because they constitute the main class of antigen presenting cells (APCs) whose primary function is to present antigens and initiate an immune response.
To generate an immune response, antigenic proteins must be processed by cells into peptides that are displayed on the cell surface by major histocompatibility complex proteins (MHCs). Circulating APCs present the peptide-MHC complexes to T cells in the draining lymph nodes, where they interact with T cell receptors, and, in conjunction with co-stimulatory signals, activate the T cells.
A variety of studies have shown that inoculation with lentiviral vectors leads to antigen presentation by DCs and strong activation of antigen specific cytotoxic T lymphocytes (CTLs; CD8+ T cells). Therefore, lentiviral vectors have been engineered for the last 10 years for gene transfer and immunotherapy applications.
The vectors routinely contain strong constitutive promoters containing enhancers, such as the CMV promoter. Michelini et al., Vaccine 27(34):4622-29 (2009); Karwacz et al., J. Virol. 83(7):30943103 (2009); Negri et al., Molecular Therapy 15(9):1716-23 (2007); and Buffa et al., J. General Virology 87:1625-1634 (2006).
Lentiviral vectors have been improved in their safety by removal of the LTR U3 sequence, resulting in “self-inactivating” vectors that are entirely devoid of viral promoter and enhancer sequences originally present within the LTRs.
The lentiviral particles, which contain lentiviral vectors, can be produced by recombinant technology upon transient transfection of cells, for example HEK 293T human cultured cells, by different DNA plasmids:
(i) a packaging plasmid, which expresses at least the Gag, Pol Rev, Tat and, in some cases, structural and enzymatic proteins necessary for the packaging of the transfer construct;
(ii) a proviral transfer plasmid, containing an expression cassette and HIV cis-acting factors necessary for packaging, reverse transcription, and integration; and
(iii) an envelope-encoding plasmid, in most cases the glycoprotein of vesicular stomatitis virus (VSV.G), a protein that allows the formation of mixed particles (pseudotypes) that can target a wide variety of cells, especially major histocompatibility (MHC) antigen-presenting cells (APCs), including DCs.
This procedure allows obtaining transient production of lentiviral particle vectors by the transfected cells. However, the lentiviral particle vectors may also be continuously produced by cells by stably inserting the packaging genes, the proviral coding DNA, and the envelope gene into the cellular genome. This allows the continuous production of lentiviral particle vectors by the cells without the need for transient transfection. Of course, a combination of these procedures can be used, with some of the DNAs/plasmids integrated into the cellular genome and others provided by transient transfection.
Non-integrating lentiviral vectors have been designed. Examples of non-integrating lentiviral vectors are provided in Coutant et al., PLOS ONE 7(11):e48644 (2102), Karwacz et al., J. Virol. 83(7):3094-3103 (2009), Negri et al., Molecular Therapy 15(9):1716-1723 (2007); Hu et al., Vaccine 28:6675-6683 (2010).
Deletion in the U3 region of the 3′ LTR of the viral promoter and enhancer sequences in self-inactivating lentiviral vectors limits the likelihood of endogenous promoter activation. These concerns with safety directly address the experiences gained from the SCID-X1 gene therapy trial carried out in 1998-1999, performed with Moloney virus-based retroviral vectors on children suffering from a rare form of X-linked (SCID-X1 gene) severe immunodeficiency disease (Cavazzana-Calvo et al., 2000, Science., 288(5466):669-72). During this trial, four of nine children developed leukemia as a result of the integration of the Moloney-derived retroviral vector at close proximity to the human LMO2 proto-oncogene (Hacein-Bey-Abina et al., 2008, J.Clin.Invest., 118(9):3132-3142). It was demonstrated that malignancy was the consequence of the proximity of the viral U3 promoter/enhancer to the LMO2 proto-oncogene. As a result, safety is a major concern for the administration of lentivectors to humans.
Promoters can contain enhancers, which are cis-acting sequences that can act as transcriptional activators at a distance. Promoters containing enhancers have been widely employed in viral derived vectors because they appear to be the most efficient for obtaining transgene strong expression in a variety of cell types (Chinnasamy et al., 2000, Hum Gene Ther 11(13):1901-9; Rouas et al., 2008, Cancer Gene Ther 9(9):715-24; Kimura et al., 2007, Mol Ther 15(7):1390-9; Gruh et al., 2008, J Gene Med 10(1) 21-32). However, given the safety issue of insertional mutagenesis, transcriptional enhancer sequences should be deleted from the lentiviral vector constructs to abolish the risk of insertional mutagenesis by enhancer proximity effect. This enhancer proximity effect is by far the most frequent mechanism of insertional mutagenesis and is the only effect described in human or animal cases of tumorigenic events after gene transfer.
Thus, there is a need to develop retroviral, particularly lentiviral vectors, which do not include viral enhancers, but still allow sufficient expression of transgenes encoding immunogenic peptide
s, if possible, as much expression as that observed when using the CMV promoter.
Recent studies has reported on the replacement of viral promoters by DC-specific promoters deriving from major histocompatibility complex class II genes (MHC class II) (Kimura et al., 2007, Mol Ther 15(7):1390-9) and dectin-2 genes (Lopes et al., 2008, J Virol 82(1):86-95). The dectin-2 gene promoter used in Lopes et al. contains a putative enhancer and an adenoviral conserved sequence (inverted terminal repeats in adenovirus promoter) (Bonkabara et al., 2001, J. Immunology, 167:6893-6900). The MHC class II gene promoter used by Kimura et al. does not contain any known enhancer.
Yet, without an enhancer, the MHC class II promoter was found not to provide sufficient transgene expression in DCs, when administered intravenously. In particular, lentiviral vectors including MHC class II promoters did not provoke an immune reaction in immunocompetent C57BL/6 mice, in contrast to the immune responses observed with CMV promoters/enhancers. Although integration and persistent transgene expression were observed after injection in mice, the lentiviral vectors transcribed through MHC class II promoters failed to stimulate an antigen-specific CD8+ cytotoxic T-lymphocyte response, even after vaccination boost. The authors of these studies therefore concluded that the use of MHC class II promoters was of interest only for applications where persistence of expression is sought as in gene replacement therapy, but not in the context of immunotherapy. Of note, MHC class II promoters are expressed poorly in most cell types.
Thus, the MHC class II promoter is not an adequate promoter for lentiviral vectors for induction of an immune response against an antigen via IV injection. Moreover, the dectin-2 promoter is expressed poorly in most cell types and appears to contain an enhancer. Thus, the dectin-2 promoter is not a good promoter for lentiviral vectors for safety reasons.
Preferably, in immunotherapy, lentiviral vectors provide effective expression of the transgene that elicits a desired specific immune response. This requires that the expression is at a high level in APCs, such as dendritic cells.
It is also preferable that the cells transduced by the lentiviral vectors are eliminated by the immune response to provide a higher degree of safety. That is, the immune response generated against the transgene can elicit an immune response in the host sufficient to eliminate the cells that are transduced by the lentiviral vectors. The elimination of transduced cells eliminates the persistence of the lentiviral vector in the host, and possible secondary effects of the vector. In order for the transduced cells to be eliminated, expression is required in non-dendritic cells at a level that allows elimination by the immune response. Thus, appropriate expression of an antigen is desirable.
At the same time, the promoter should maximize immune stimulation through the key cells (i.e., dendritic cells) involved in the activation of naïve and memory T cells, and should minimize the risk of insertional mutagenesis and genotoxicity in stem cells, leading to malignancies. Thus, the promoter should have sufficiently high activity in dendritic and other cells, but not contain an enhancer. Based on these criteria, viral promoters, such as the CMV promoter, are not ideal because of the presence of strong enhancers. These criteria are summarized as follows:                1. high expression in antigen presenting cells, including dendritic cells, to induce maximal immune responses;        2. expression in other transduced cell types sufficient for elimination by the induced immune response; and        3. lack of an enhancer element to avoid insertional effects.        
Human T Lymphotrophic Virus type 1 (HTLV-1) is the etiologic agent of Adult T-cell Leukemia/Lymphoma (ATL) (Poiesz, et al., 1980, Proc. Natl. Acad. Sci. USA, 77(12):7415-7419; Yoshida, et al, 1982, Proc. Natl. Acad. Sci. USA, 79:2031-2035). HTLV-1 is also causatively associated with other pathologies for which there is no cure or effective treatment: myelopathy/tropical spastic paraparesis (HAM/TSP), an inflammatory chronic meningomyelitis of the grey and white matter in the spinal cord with perivascular demyelination and axonal degeneration; and uveitis and autoimmune conditions. One agent, HTLV-1, is thus responsible of at least two diseases (ATL and HAM/TSP); infected individuals never develop both.
20 million of individuals worldwide are estimated to be infected by HTLV-1, with determined endemic areas (i.e. Japan, some African countries, the Caribbean islands and Central and South America) and different virus subtypes whose predominant, cosmopolitan subtype A, shows a low genetic variability (Gessain and Cassar, 2012, Front. Microbiol, 3:388). However, the estimated lifetime risk for HTLV-1 infected people to develop ATL and inflammatory chronic diseases (HAM/TSP) is lower than 5%, usually 20-30 years after the onset of infection. The majority of infected people remaining asymptomatic carriers, deciphering HTLV-1 pathogenesis mechanisms is a matter of concern.
ATL is a malignant lymphoproliferative disease which has been classified into four subtypes: smoldering, chronic, lymphoma and acute (Shimoyama, 1991, Br. J. Haematology, 79(3):428-437). Classification is performed according to the following criteria: lymphocyte counts, percentage of atypical lymphocytes, lactate dehydrogenase (LDH) level, calcium level and skin lesions. A fifth state is also sometimes referred to, “pre-ATL”, that is characterized by an asymptomatic disease with presence of abnormal peripheral blood lymphocytes with typical ATL morphology. ATL cells have indeed a so-called “flower cells” aspect with condensed chromatin, small or absent nuclei and agranular and basophil cytoplasm.
Patients developing ATL usually experience lymphadenopathy, fever, skin lesions, leucocytosis and hepatosplenomegaly. For indolent ATL subtype (i.e. chronic and smoldering subtypes), the median survival time is approximately 4 years (Takasaki, et al, 2010, Blood, 115(22): 4337-4343): 2 to 5 years for the chronic subtype and approximately 3 years for the smoldering one. However, the median survival time for patients with aggressive subtype (i.e. acute, lymphoma, or unfavorable chronic type ATL) decreased to 5 to 13 months even in prospective trials employing multi-agents chemotherapy.
Prognostic factors are advanced performance status, high calcium level, high lactate dehydrogenase level, age (more than 40 years old) and more than three involved lesions. Of note, individuals developing ATL are more prone to develop develop opportunistic infections (Oliere et al, 2011, Cytokine Growth Factor Rev, 22(4): 197-210).
Treatment of ATL is dependent on the disease subtype (Oliere et al, 2011, Cytokine Growth Factor Rev, 22(4): 197-210). However, therapeutic options are very limited and available therapies only delay the time to relapse.
Although the mechanism of action remains unclear, studies assessing efficacy of the combination of azidothymidine (AZT, Zidovudine) with interferon-alpha gave encouraging results. A meta-analysis of the trials assessing AZT plus interferon-alpha showed that 5-year overall survival reached 46%, a value never reported for any other experimental ATL treatment (Bazarbachi et al, 2010, J. Clin. Oncol., 28(27):4177-4183). Survival benefit was observed especially in the leukemic ATL subtypes (acute, chronic and smoldering) and when the treatment was administered as first-line. However, treatment with AZT/interferon alpha has many side effects and it is a lifelong treatment without interruption.
Preliminary results of adding arsenic trioxide to this combination therapy suggested that it might be beneficial as consolidation therapy and is worth being investigated further (Kchour, et al, 2013, Retrovirology, 10:91).
In Japan, a Phase III clinical trial showed that the mLSG15 regimen that consisted of sequential administration of three drugs associations-VCAP (vincristine, cyclophosphamide, doxorubicin, prednisolone), AMP (doxorubicine, ranimustine, prednisolone) and VECP (vindesine, etoposide, carboplatin, prednisolone)—was superior to biweekly CHOP (cyclophosphamide-hydroxydaunorubicine-oncovin-prednisone) in newly diagnosed acute, lymphoma and unfavourable chronic ATL with median progression-free survival of 7.0 months and overall survival of 12.7 months (Tsukasaki et al, 2007, J. Clin. Oncol., 25(34): 5458-5464).
To date, it is still up to the clinicians to decide whether, depending on the individual benefice risk ratio, to use CHOP or VCAP-AMP-CECP when the treatment option chosen is multiple agents chemotherapy.
During a Phase II clinical trial performed on patients who experienced relapsed or refractory ATL, a humanized monoclonal anti-CC chemokine receptor 4 (CCR4) antibody was shown to be effective for ATL, especially in the acute subtype disease. 27 patients enrolled in the study were treated with the antibody, mogamulizumab. Among the 26 patients who were evaluable, 13 achieved an objective response and among them, 8 a complete response (Ishida et al, 2012, J. Clin. Oncol., 30(8):837-842). In March 2012, mogamulizumab was approved in Japan for the treatment of relapsed or refractory ATL (brand name POTELIOGO®). Post-marketing surveillance reports several serious skin-related adverse events including Stevens-Johnson syndrome (one of them fatal), urging the need to better understand the optimal treatment strategy with mogamulizumab (Ishida et al, 2013, Cancer Sci., 104(5): 647-650). Recently, association of mogamulizumab provided additional progression free survival (PFS) when added to mLSG15 compared to mLSG15 alone, in patients with acute, lymphoma and unfavourable chronic ATL.
Other monoclonal antibodies are being investigated, such as antibody directed against the CD25 which has been assessed in clinical trials, alone or coupled to yttrium-90. An anti-transferrin receptor antibody gave also encouraging results in preclinical stage.
Promising results have been obtained with allogenic hematopoietic stem cell transplantation (allo HSCT) as a curative treatment of ATL. Though, the number of patients who might benefit from this option is very limited (patient developing ATL are usually old and therefore clinicians are reluctant to perform this operation; in addition, finding a compatible donor can prove difficult) (Obama et al, 1999, Int. J. Hematol., 69(3):203-205). Besides, in the few patients eligible for allo HSCT, 30% of patients develop Graft Versus Host disease with severe side effects or leading to death, about 30% of patients relapse and only approximately 30% of patients are cured.
One interesting study reported the successful treatment of a patient with mogamulizumab followed by allo HSCT after treatment failure with chemotherapy (Motohashi et al, 2013, Int. J. Hematol, 98(2):258-260).
Hence, although some clinical trials have given encouraging results by increasing the response rates, most of the therapies failed to achieve a significant impact on long-term survival. Moreover, the tested treatments are mainly aggressive ones.
New drugs, already approved or not for treatment of other T-cell lymphomas, are being assessed in ATL patients. These are, for example, the vorinostat and romidepsin histone deacetylase inhibitors (HDAC), FDA-approved for the treatment of relapsed and refractory cutaneous T-cell lymphoma or alemtuzumab, and an anti-CD52 antibody, approved for the treatment of chronic lymphoid leukemia.
New treatments for ATL patients with better overall survival impact, low side effects and possibly not lifelong treatment, either in aggressive or indolent forms of the disease, are desperately searched for.
One hypothesis to explain the long latency period before asymptomatic HTLV-1 carriers (AC) develop ATL is a balance between host immune response and HTLV-1 genome expression (Yoshida, 2010, Proc. Jpn Acad. Ser. B. Phys. Biol. Sci., 86(2): 117-130).
Indeed, several observations and experiments point to a crucial role of host immune system in controlling HTLV-1 spread and the development of HTLV-1 related diseases in infected patients. Among them, in animal model (rat), vertical transmission of HTLV-1 by breastfeeding leads to immunotolerance causing a higher risk for ATL (Hasegawa et al, 2003, J. Virol., 77(5):2956-2963; Komori et al, 2006, J. Virol., 80(15):7375-7381). However, subcutaneous injection of HTLV-1 infected rat cells before oral infection, prevented ATL appearance. Others have reported development of ATL in asymptomatic carriers (AC) treated with immunosuppressants after liver transplant (Kawano et al, 2006, Transplantation, 82(6):840-843; Suzuki et al, 2006, Int. J. Hematol., 83(5):429-432). Moreover, an increase of anti-HTLV-1 immune response in ATL patients treated with allo-HSCT has been observed and called Graft Versus Leukemia (GVL) effect, leading to patients' remission (Harashima et al, 2004, Cancer Res., 64(1):391-399). Furthermore, low anti-HTLV-1 immune responses in ATL have been described, which could favor the initiation and progression of the disease in patients (Kannagi et al, 2011, Cancer Sci., 102(4):670-676; Kannagi et al, 2012, Front. Microbiol., 3:323).
In vitro experiments demonstrated that the CTLs specific to HTLV-1 recognized mainly Tax and to a lesser extent, the envelope, polymerase, p12, p30 and HBZ (reviewed in (Kannagi et al, 2012, Front. Microbiol, 3:323)). CD8+ T cells originating from AC and ATL patients have been assessed for frequency, diversity and polyfunctionality; results demonstrated an impaired response in these three parameters in ATL versus AC patients (Kozako et al, 2006, J. Immunol., 177(8): 5718-5726; Manuel et al, 2013, J. Clin. Immunol., 33(7):1223-1239).
These, and other studies, demonstrate that specific HTLV-1 cellular immune response is dramatically impaired in patients who have developed ATL. Hence, therapy which aims at stimulate cellular immune response against HTLV-1 infected cells could be an appropriate therapeutic option to treat ATL.
Preclinical studies have already demonstrated the efficiency of a vaccine against the HTLV-1 viral protein Tax in the treatment of ATL phenotype in animal models. Indeed, in a rat model of ATL phenotype (Ohashi et al, 1999, J. Virol., 73(7):6031-6040), in vivo vaccination with Tax DNA induced the stimulation of Tax specific CTLs which are able to lyse HTLV-1 cells in vitro. An adoptive transfer of these CTLs simultaneously with injection of HTLV-1 infected cells inhibits tumor growth in vivo ((Ohashi et al, 2000, J. Virol., 74(20): 9610-9616).
In another study, engraftment of ATL CD4+ cells from acute or chronic ATL subtypes patients leads to ATL like phenotype in NOG mice. Simultaneous injection of CTL from patients, in vitro stimulated with Tax peptides, leads to a decrease in ATL lesions due to an infiltration of CTL in the tumor site, which recognize and kill HTLV-1 tumoral cells Masaki et al, 2013, J. Immunol., 191(1):135-144).
Recently, a clinical trial phase I of a therapeutic vaccine using autologous dendritic cells pulsed with peptides derived from viral protein Tax, as a treatment of ATL, showed preliminary encouraging results: reduction of the proviral load and reduction of the size of the surface lymph nodes (Suehiro, et al, 2013, abstract book from the 16th International Conference on Human Retrovirology: HTLV and Related Viruses). More impressively, 1 of the 2 patients who completed the study achieved a partial remission and the other one has a stable disease without severe side effects.
All these data confirm that the stimulation of the cellular immune response against HTLV-1 cells could be a strong therapeutic option to treat ATL patients.
The first disadvantage of ex vivo peptidic vaccination used in the only clinical trial testing a vaccination against HTLV-1 is the selection of patients eligible for treatment according to their HLA haplotype. Secondly, ex vivo maturation of autologous DCs requires purification steps from PBMCs of patients, leading to repeated depletion of circulated mononuclear cells. In a pathological context, this could be detrimental for the immune system of patients. Moreover, purification of autologous DCs is very expensive and within technical challenge to get good performance (as an example, see PROVENGE® vaccine from Dendreon in prostate cancer treatment (Huber et al, 2012, J. Natl. Cancer Inst., 104(4):273-279)).
Thus, a need exists in the art for improved vectors and methods for treatment of HTLV-1 in humans. The present invention fulfills these needs in the art.